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The flow behaviour of a polymer solution has been reported to depend on the composition and stability of the composite suspensions [211]. Viscosity was therefore analyzed for both polymer (CS) and polymer-ceramic solutions (CS/β-TCP) under varied shear stress. All the solutions have shown non-Newtonian behaviour with shear thinning property. Figure 5.2 depicts the shear thinning ability of pure and composite [CS/micro β-TCP (80:20), CS/nano β- TCP (80:20)] solutions. CS solution has shown lower viscosity (1.21 Pa s) in comparison to the composite solutions with varied micron (1.31-1.99 Pa s) and nano sized β-TCP content (1.25-1.36 Pa s). It is further observed that the increase in β-TCP content increases the

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viscosity of CS/β-TCP composite solution in a linear fashion. The viscosity of all other solutions with different β-TCP ratios is shown in Table 5.1. As indicated, the decrease of particle size decreases the viscosity of suspension as reported earlier [250]. The homogeneity of composite solution is observed to be decreased as TCP concentration increases. As a result, the layers in the composite solutions begun to show restricted movement cumulating in increased viscosity. This alteration in viscosity has an impact on pore size of developed scaffolds as reported earlier [53].

Figure 5.2: Rheological behaviour of CS, CS/micro β-TCP (80:20) and CS/nano β-TCP (80:20) composite solutions. All the solutions show non Newtonian behaviour with shear thinning property. CS/micro β-TCP and CS/nano β-TCP scaffolds are represented as CS/µ β-TCP and CS/n β-TCP in the figure respectively. [Data with other ratio of CS and β-TCP are not shown]

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Table 5.1: Viscosity of CS, composite scaffolds with varied content of β-TCP

S.No Sample Viscosity (Pa s)

1 CS 1.214 2 CS/µ β-TCP (90:10) 1.317 3 CS/µ β-TCP (80:20) 1.609 4 CS/µ β-TCP (70:30) 1.722 5 CS/µ β-TCP(60:40) 1.860 6 CS/µ β-TCP(50:50) 1.991 7 CS/n β-TCP (90:10) 1.256 8 CS/n β-TCP (80:20) 1.277 9 CS/n β-TCP (70:30) 1.313 10 CS/n β-TCP (60:40) 1.342 11 CS/n β-TCP (50:50) 1.368 5.1.3 Preparation of scaffolds

3D porous chitosan and its composite scaffolds with varying amount of micro and nano size β-TCP were prepared by freeze-gelation method. The scaffolds are shown in Figure 5.3. The scaffolds are designated as CS (pure chitosan), CS/micro β-TCP (composite scaffold with micro size β-TCP) and CS/n β-TCP (composite scaffold with nano size β-TCP).

Figure 5.3 Developed pure CS scaffolds (a), CS/nano β-TCP composite scaffolds (b, c) and CS/micro β-TCP freeze-gelled composite scaffolds (d)

52 5.1.4 Morphology and pore size

Adequate pore size and interconnectivity of pores in scaffolds are vital factors for the diffusion of oxygen and nutrients to the cells and foster transfer of metabolic wastes [102]. SEM images were taken to visualize these features. As observed, pure CS scaffold shows porous and excellent interconnected network of open pore microstructures [Figure 5.4-(a)] with a pore size range of 61-171 μm. Similar morphology and pore structure are also achieved with composite scaffolds. However, the pore diameter of CS scaffolds is found to decrease with increase in β-TCP content. This may be attributed to a slight increase in viscosity of composite solutions with increase in β-TCP content, which leads to restricted movement of ice crystal during freezing as reported earlier [54]. The ice crystal mobility responsible for pore diameter is reduced in high viscous solutions and hence a decrease in pore size of composite scaffolds is observed as compared to pure CS scaffolds. Similar observation of decrease in pore size was reported earlier when β-TCP was incorporated in various weight ratios into CS/gelatin 3D composite matrices developed for bone tissue regeneration [53]. The other reason may be due to the loss of structural integrity of scaffolds at higher content of β- TCP resulting in irregularity in the pores and thus decrease in pore size [54]. Similar findings related to decrease in pore size of CS scaffolds reported earlier by incorporating bio-ceramics such as HAp, β-TCP and wollastonite [54, 68-69]. As observed, β-TCP is found to be more or less uniformly distributed over the scaffold surface and on the pore walls. Figure 5.4 illustrates that there is no significant difference observed between pore size of nano β-TCP and pure CS scaffolds. On the contrary, scaffolds with varied micro β-TCP composite scaffolds show smaller pore size in comparison to pure CS scaffolds as shown in Figure 5.4 (b - f). The pore size range obtained with varied micro β-TCP content is 45-165 μm. However, after a certain limit of micro β-TCP content (>30% wt), the distribution of β-TCP is found to be non uniform due to the formation of agglomerates along the periphery of the pores as shown in Figure 5.4 (A-b) and Figure 5.4 (B) Whereas with CS/nano β-TCP composite scaffolds, the pores are found to be regular [Figure 5.4 (A - c) and Figure 5.4 (C)] and β-TCP is uniformly distributed over the entire scaffold surface and in the pore walls. The pore size range obtained with CS/nano β-TCP scaffold is 51-168 μm. The pore size range of

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developed scaffolds with varied ratios of micro and nano β-TCP were measured and depicted in Table 5.2. Published reports suggest that the pore size of scaffolds should be in the range of 50-300 μm for bone tissue engineering applications and results obtained matches with the reported pore size range [134]. Thus the developed freeze-gelled CS and CS/β-TCP composite scaffolds have the required morphological necessity in terms of pore size and interconnecting pore networks for proper cell growth and proliferation of osteoblasts.

Figure 5.4: SEM images of the prepared freeze-gelled scaffolds. Open porous microstructure with interconnectivity is evident in pure CS and CS/β-TCP composite scaffolds. Decrease in pore size of composite scaffolds with increase in β-TCP content is noticed

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Figure 5.4 (A): SEM images of scaffolds at higher magnification. Pure CS scaffold (a); agglomerates of micro β-TCP are represented with white colour arrow marks (b); uniform distribution of nano β-TCP is represented with white colour arrow marks in (c)

Figure 5.4 (B): SEM images of CS/β-TCP scaffolds at higher magnification. Agglomerates of micro β-TCP are represented with white colour arrow marks

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Figure 5.4 (C): SEM images of CS/β-TCP scaffolds at higher magnification. Uniform distribution of nano β-TCP is represented with white colour arrow marks

5.1.5 Porosity

The porosity of scaffold is an important factor that decides not only the transport of nutrients required for cell growth but also cell infiltration and migration [5, 117]. Table 5.2 shows the % porosity of the developed scaffolds. As indicated, the porosity of CS scaffold (86 ± 3.21) is decreased with increase in both nano and micro β-TCP content. The corresponding % porosity of CS/micro β-TCP and CS/nano β-TCP composite scaffolds are 80.7 ± 2.33 to 74.2 ± 5.2 and 84.6 ± 2.65 to 78.3 ± 2.9 respectively. Furthermore, the decrease in porosity is slightly higher in case of scaffolds with micro β-TCP than scaffolds loaded with nano β-TCP. The overall trend of porosity is CS>nano β-TCP>micro β-TCP. However, the porosity of the composite scaffolds is still high enough for bone tissue engineering applications. This phenomenon of decrease in porosity may be attributed to the gradual increase in the density of β-TCP in CS matrix as reported earlier [54]. The porosity result has followed the same trend as pore size and the result obtained in our study is in good agreement with the results reported earlier when wollastonite and HAp were used for the development of composite CS scaffolds [54, 119].

56 5.1.6 Phase analysis

X-ray diffraction pattern was used to analyze the phase changes, if any occured in CS based composite scaffolds. A broad dome shaped curve is shown with pure CS scaffold representing its amorphous nature as shown in Figure 5.5 [38], whereas in composite scaffolds, the CS peak is completely dominated by the incorporation of ceramic which is attributed to high crystalline nature of β-TCP [36]. The broad, dome-shaped and low-intensity peaks at 2θ = 9.16° and 20.6° correspond to pure CS and that of β-TCP are at 2θ = 31.7°, 32.1° and 39.7° confirming the presence of β-TCP in the composite scaffold. Furthermore, the effect of nano β-TCP is higher in dominating the CS peaks at 2θ = 20.6° as compared to micro β-TCP. Diffractograms of composite scaffolds with varied ratios of micro and nano β-TCP are shown in Figure 5.6 (C-A and B). As indicated, no significant change in peak intensity of β-TCP is observed with varied size of β-TCP. Further, the crystallite size of β-TCP particles in micro and nano size in composite scaffold were calculated using XRD peaks of Figure 5.5 (B). The corresponding values are 30.8nm and 10.82nm respectively. Thus it is established that the increase in crystallinity of CS/β-TCP composite scaffold is due to β-TCP as indicated from Figure 5.5 and 5.6.

Figure 5.5 (A): XRD pattern of pure CS, CS/micro β-TCP (90:10) and CS/nano β-TCP (90:10) composite scaffolds. Peaks at 2θ=9.16° and 20.6° denotes CS where as twin peaks at 2θ=31.7° and 32.1° denote β-TCP. CS peak at 2θ= 20.6° is dominated by the incorporation of β-TCP

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Figure 5.5 (B): XRD pattern of CS/micro β-TCP and CS/nano β-TCP (90:10) composite scaffolds.Crystallite size of micro and nano β-TCP is 30.8nm and 10.82nm respectively

Figure 5.6 (C): XRD patterns of freeze-gelled composite scaffolds consisting of varying ratios of chitosan and β-TCP. Extent of decrease in amorphous nature of CS with respect to size of β-TCP is well represented. Nano sized β-TCP has completely dominated the amorphous nature of CS indicating higher crystallinity to composite scaffolds as compared to scaffolds with micro β-TCP as denoted with black colour arrow marks. The XRD peaks of composite scaffolds are compared with XRD peaks of pure CS peaks

58 5.1.7 Functional analysis

An infrared spectrum represents the characteristics of a material with absorption bands. Thus infrared spectroscopy can provide a positive identification of every different constituent present in the composite matrices [154]. FT-IR analysis of CS and CS/β-TCP composite scaffolds was therefore, done to evaluate the interaction between the individual components present in the scaffolds. The bands generated as a result of chemical interactions is shown in Figure 5.7. Pure CS scaffold shows bands around 897 and 1154 cm-1 of assigned saccharide structure and the characteristic bands of N–H bending (amide-I) of CS is observed at 1560 cm-1 [70]. The typical band range of tetrahedral anions is 790–1190 cm-1. Our analysis has demonstrated PO4-3 bands at 1040, 1122, 610, and 551 cm–1 fall within the reported range [211].Reports suggest the shifting and formation of new bands due to the interaction of ceramic material with the polymer functional groups [38]. The bands ranging from 1560-1587 cm-1 N-H bending has been shifted to 1556-1573 cm-1 which may correspond to the interaction of β-TCP with CS. The bands around 1000-1300 cm-1

are due to the interaction of phosphate with oxygen and nitrogen of CS [130]. IR pattern of composite scaffolds with varying content of β-TCP are not shown as no significant variation between the obtained peaks is observed.

Figure 5.7: FT-IR spectra of CS and composite scaffolds. Bands ranging from 1560-1587 cm-1 corresponds to N-H bending has been shifted to 1556-1573 cm-1 due to the interaction of P=O with CS

59 5.1.8 Compressive strength

The scaffolds must possess enough strength to withstand the force exerted by the neo tissue formed under in-vivo handling of implanted scaffold [212]. The stress strain curve and the corresponding compressive strength of the developed porous CS and CS/β-TCP composite scaffolds are shown in Figure 5.8 (a) and (b) respectively. It is clear from Figure 5.8 (b) that, the compressive strength (0.19 ± 0.05) of CS scaffold is remarkably increased with the addition of both types and content of β-TCP. The increase in compressive strength is due to reinforcing effect of bio-ceramic. The enhanced mechanical strength of polymeric scaffolds by the incorporation of bio-ceramic has also been reported earlier[162]. Further, the compressive strength of CS/micro β-TCP composite scaffolds is increased with increase in β- TCP content which is due to the good dispersion of the ceramic material into the CS polymer matrix that helped the stress to spread over a larger area of the composite scaffold surface. However, a decline in compressive strength is observed when the β-TCP content was over 30%. This decrease in compressive strength at higher β-TCP content is due to the agglomeration of β-TCP observed in CS matrix (Figure 5.5) that led the stress to be concentrated at the interface of the polymer-ceramics. When the particle size is in nano range, the reinforcement effect is found to be better in achieving higher compressive strength [213]. A linear relationship of compressive strength is observed with CS composites loaded with nano β-TCP (upto 40 wt%) and a slight decrease in compressive strength is observed with further increase in β-TCP content. This may be due to the decreased ability of scaffold to further reinforce the ceramic material into CS polymer matrix. More precisely, the compressive strength of scaffolds with nano β-TCP has been increased steadily upto 40% [CS/nano β-TCP 60:40-2.67 ± 0.21 MPa] and then a slight decrease in strength is observed [CS/nano β-TCP 50:50-2.55 ± 0.15 MPa] which represents the desired compressive strength of typical cancellous bone 2-10 MPa [214]. The compressive strength of all the developed scaffolds with varying micro and nano β-TCP content is depicted in Table 5.2. The high surface-to-volume ratio of nano size particle results in improved mechanical properties of bio- ceramics as reported earlier with β-TCP, wollstonite, HAp and bioglass [4, 69, 149, 187].A similar increase in compressive strength of CS gel and 3-D CS scaffolds has also been

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reported and the strength was increased to 0.8 and 1.8 MPa respectively by incorporating β- TCP [10, 53].

Figure 5.8 (a): Stress-Strain plot of CS and CS/β-TCP composite scaffolds

Figure 5.8 (b): Compressive strength of CS and CS/β-TCP composite scaffolds with different weight ratios of CS and β-TCP. Compressive strength of CS/ micro β-TCP composite scaffold increases with increase in β-TCP content upto 70:30 CS/ β-TCP ratio beyond which a decline trend is observed due to the agglomeration of β-TCP in the polymer matrix. Increase of nano β-TCP content has improved compressive strength of composite scaffold upto 40%

61 5.1.9 Swelling behaviour

The swelling behaviour is one of the key factors of a scaffold determining its ability to qualify for in-vitro cell culture study. Swelling enables the cells to utilize the internal region of scaffolds upto maximum possible extent [234-235]. The trend obtained with swelling behaviour of CS and CS/β-TCP composite scaffolds is represented in Figure 5.9. The % swelling of all the developed scaffolds with varying β-TCP content is shown in Table 5.2. Among the developed scaffolds, pure CS scaffold shows a higher swelling percentage (~326%) than any of the CS/β-TCP composite scaffolds. The swelling ratio is observed to be decreased with increase in ceramic content. The decrease in % swelling of composite scaffolds is caused by the incorporation of β-TCP in the polymer matrix which ultimately leads to a decrease in diffusion of water as reported earlier when HAp was used as filler in CS [54]. The composite scaffolds with nano β-TCP shows higher swelling rate (~190 to 275%) as compared to scaffolds loaded with micro β-TCP (~155 to 212%) which may be due to the difference in distribution ability of micro and nano sized filler [38]. The other reason for the difference in swelling behaviour of composite scaffolds is may be attributed to gradation in pore size and porosity [38].

Figure 5.9: Swelling behaviour of CS and CS/β-TCP composite scaffolds. Pure CS scaffold shows a higher swelling than composite scaffolds. The swelling ratio decreases with increase in ceramic content that implies that the ceramic content has a direct influence on the diffusion of water into the scaffold matrix

62 5.1.10 Measured contact angle

The water contact angle is determined to assess the hydrophilicity characteristics of the scaffold surface which is important for various cellular responses as reported earlier [215]. Therefore, the contact angle of the best composite scaffolds selected based on high mechanical strength was measured and compared with pure CS scaffold. There is no significant change in contact angle is observed between pure CS scaffold (51.2 ± 0.8°) and CS/nano β-TCP (60:40) [51.9 ± 1.1°] which represents their similar hydrophilic characteristics. However, a slightly lower hydrophilicity characteristic is shown by CS/micro β-TCP (70:30) [53.1 ± 0.5°] scaffold which may be attributed to micron size of the ceramic particle which restricted the flow of water through the scaffold architecture. Thus it has been demonstrated the superior surface property of CS composite reinforced with nano β-TCP is favourable for bone tissue engineering application than the composite made with micro sized β-TCP.